Performance of chlorophyll a fluorescence parameters in Lemna minor under heavy metal stress induced by various concentration of copper

The objective of the present investigation was to understand the efficacy of chlorophyll fluorescence analysis and to identify the specific photosynthetic parameters for early and rapid detection of Cu-induced HM-stress in plants. Aquatic angiosperm Lemna minor was exposed to various concentrations (0–40 µM) of Cu. We observed that the FV/FO (Efficiency of the water-splitting complex on the donor side of PSII), quantum yield for electron transport, and quantum yield of primary photochemistry were decreased however, dissipated quantum yield was increased with Cu concentration. ABS/CSM, TRO/CSM, ETO/CSM and maximum quantum yield were displayed the dose–response relationship under Cu stress. Performance indexes were increased initially due to the beneficial effects of Cu at lower concentration while decreased significantly (p ≤ 0.05) at highest concentration of Cu. The outcomes of the present research revealed that the ChlF analysis is very sensitive tool that can be used to determine the toxicity of heavy metals in plants.


Scientific Reports
| (2022) 12:10620 | https://doi.org/10.1038/s41598-022-14985-2 www.nature.com/scientificreports/ the portion of absorbed energy which is not utilised to drive photosynthesis 23,[41][42][43][44][45][46] . ChlF measurement provides information about changes in photosynthetic efficiency and heat dissipation 47,48 . It is an incredibly simple, noninvasive, extremely sensitive, rapid, and accurate method and providing a quantitative assessment of oxygenic photosynthesis 37 . Plants exposed to HM ions disrupt photosynthesis as a result of a single or cumulative event of HM interaction with protein which increase the rate of ROS generation and which replaces essential kations in protein active centers 28 . Some HM ions, for example, Cu, Hg, Cd, Zn, or Ni can replace the core Mg ion in chlorophyll molecules, resulting in chlorophyll-metal complexes and a reduction in PSII quantum efficiency [49][50][51] . Apart from evaluating specific parameters, of which the F V /F O and F V /F M are the most well-known and extensively utilised, the interpretation of double normalised curves using the JIP test is becoming increasingly popular in environmental research practices 42 . Plots are formed using data collected at a high sampling rate within a second of the dark-adapted leaf being exposed to light, as the independent variable on a logarithmic timeline. On such plots, inflection points (J-I-P) are noticed when the recorded fluorescence increases which provide the foundation for inferences regarding the photosynthetic apparatus' structure and function. the O-J-I-P transient is prime source of observed variations in the efficiency of the chlorophyll antenna in capturing light energy and transfer to plastoquinone Q A (the electron acceptor) is the only limitation of photochemical conversion in PSII 52,53 . Even though, ChlF there are years of in-depth expertise, valid interpretations of ChlF data still require more research 54 . ChlF measurement has become a simple, effective, and dependable technique for outdoor environmental research to improve knowledge and current technology 42,43,45,[55][56][57][58][59] .
HM, pollution is becoming more prevalent in the environment, demanding rapid and effective solutions for metal remediation. The use of metal-accumulating plants for remediation has recently given rise to a new technology known as phytoremediation 60 . An ideal hyperaccumulator plant species must meet two requirements for this technology to be viable are HM tolerance and accumulation. Consequently, a better knowledge of the metal tolerance mechanism(s) is critical for the development of effective phytoremediation techniques 61 . The chlorophyll a fluorescence has long been used to measure the effects of environmental stress on plants, because they provide a quick approach to determine injury in the absence of visual signs 62,63 . Therefore, the ability to identify the toxic effects before any morphological symptoms can be seen makes phytoremediation an extremely effective method for identifying metal-tolerant plants.
Duckweeds have high potential to grow under HM stress because of their potential to bioremeidte HMs through either by rhizofilteration or phytotransformation. Therefore, besides use in bioremediations, duckweeds serve a rich source of essential HMs such Cu and Zn for improving feed efficiency of animals 64 .
Chlorophyll (Chl) a fluorescence signals have become one of the potent indicators for early detection of HMs in soil and aquatic bodies 25,65,66 . In the present study, we used the chlorophyll (Chl) a fluorescence transient to investigate the effects of HM stress induced by various concentration of Cu in L. minor plants grown in a nutrient medium.

Materials and methods
Plant material and growth condition. L. minor plants were collected from the region of Ayad river located at Udaipur, India (24° 35′ 14.97′′ N, 73° 42′ 38.75′′ E) (As per the Biological Diversity act, 2002 of National Biodiversity Authority of India, the Indian researchers neither require prior approval nor need to give prior intimation to SBB for obtaining biological resource for conducting research 67 ). The plant was identified by Dr. Vineet Soni based on the morphological characteristics (oval shaped fronds, 2-5 fronds remained together, presence of three nerves in each frond and cylindrical root sheath with two lateral wings). The collected fronds (stock culture) were maintained in plastic (PVC) aquariums in Jacob culture media as per the OECD guideline of 2002 68  Cu exposure. For the ChlF experiment ~ 30 two or three-fronded, healthy plants (300 mg) were taken from stock culture and transferred to glass bottle containing 250 mL of growth medium without EDTA and exposed to various concentrations of CuSO 4 ·5 H 2 O (Sigma Aldrich, C8027, ≥ 98%) (0, 10, 20, 30, and 40 μM) for 24 h. The metal exposure experiments were performed according to procedure described by Teisseire and Guy using EDTA free growth medium since it is a chelating agent and alter the metal adsorption process in plants (can increase the bioavailability of metal) 70 . Control plants were grown under both EDTA and Cu free growth medium. The experiment glass bottles were placed in a controlled environment as described above.
Chlorophyll a fluorescence transient. ChlF was measured using a plant efficiency analyser (Handy PEA fluorimeter, Hansatech instruments Ltd. England). Before measurement fronds were dark-adapted for 50-60 min at 26 °C. Thereafter, ChlF signals were analysed with the Biolyzer v.3.0.6 software (developed by Laboratory of Bioenergetics, University of Geneva, Switzerland). The experiments were done in six replicates and repeated three times to ensure the results. JIP-test method has been developed by which several selected phenomenological and biophysical parameters quantifying the PSII and PSI behaviors are calculated. Several parameters can be derived from the polyphasic ChlF rise (OJIP curve) that provide information about photo-Statistical analysis. Statistical analysis was performed using analysis of variance (ANOVA), followed by a Tukey HSD test (p = 0.05) using XLSTAT 2020. Only significant values (p ≤ 0.05) of measurements are presented in figures. The heat map was prepared by normalizing the values and bringing them all to a range between 1 and 100 to provide an unbiased color code. Three color code combination of red for high (100%), yellow for medium (50%), and green for the lowest value (1%) was used to represent the heat map. The MS excel and CorelDraw software were used for calculation and designing of the heatmap. In addition, a principal component analysis (PCA) was conducted by eigenvalue decomposition of a data correlation matrix using OriginPro 2016. PCA was applied to find the patterns of the fluorescence parameter and variations in the experimental data. The 48-h lethal dose (LD 50 and LD 90 ) was determine by Probit Analysis using SPSS (22.0). Comparision of mortility ratios between experimental and control groups in the deferent concentrations was performed with Chi-square testing.

Results
Cu stress significantly altered the growth and productivity of L. minor through the modulation of the photosynthetic process. In the present studies, impacts of Cu-induced HM stress on ChlF kinetics, specific energy fluxes, phenomenological energy fluxes, and performance indexes were studied in L. minor.
ChlF rise. ChlF rise of L. minor was measured after 24 h of Cu treatment and a typical OJIP induction curve was displayed when plotted on the logarithm time scale (Fig. 1). With increasing the Cu concentration, the fluorescence yield at various intermediary steps, such as J, I, and P was reduced. In control plants, two intermediate peaks F J (chlorophyll fluorescence at 2 ms) and F I (chlorophyll fluorescence at 300 ms) were formed between F O and F M , ChlF increased continuously from initial (F O ) to maximal (F M ) fluorescence intensity in L. minor growing under control conditions. HM induced reduction in PSII photochemistry and electron transport activity were severe at the highest concentration of Cu.  The relative variable fluorescence at 2 ms (J step) is denoted as V J which is the measure of the fraction of primary quinone electron acceptor of PSII in its reduced state [Q A − /Q A(total) ] 74 . At the lower level of HM stress, a slight reduction in the value of V J was observed but with increasing the metal concentration to a high level the V J value was increased to 224.74% of control (Figs. 2A, 3).
Complimentary Area (S M ) is also an important parameter that is directly proportional to the number of reduction and oxidation of one Q A molecule during the fast OJIP transient or number of electrons passing through the electron transport chain 75 . The turnover number (N) is represented as the number of times Q A becomes reduced and re-oxidized another time, till the F M (Maximum fluorescence intensity) is reached [76][77][78][79] . At severe Cu stress the increased value of turnover number (N) value was recorded (145.40% of control) which was also represented by PSI cyclic electron transport as photoprotection ( Fig. 2A). The increased values of S M in L. minor Table 2. Abbreviations, formulas, and definitions of the JIP-test parameters.
Turnover number (expresses how many times Q A is reduced in the time interval from 0 to tF M ) 81 Relative variable fluorescence at t = 2 ms 81 Performance indexes Performance index for energy conservation from photons absorbed by PSII until the reduction of intersystem electron acceptors 74, 81, 110     (Fig. 5). Electron transport efficiency of plants was found tolerant to Cu-induced HM stress. A high concentration of Cu (40 µM) extremely reduced the electron transfer system in thylakoid membranes (Fig. 5). DI O /CS M was increased significantly (p ≤ 0.05) at 30 µM Cu treatment and after that decreased slightly).

K P and K N .
De-excitation rate constants for nonphotochemical reaction (K N ) increased under Cu stress and at severe stress conditions K N value approaches 198.46% of the control (Figs. 2C, 3). While de-excitation rate constants for photochemical reaction (K P ) lowered slightly (86.48% of control) at 40 µM Cu concentration.
Performance index. Overall effects of Cu-induced HM stress on various photosynthetic parameters are presented in the form of a radar plot (Fig. 2). To analyze the effects of Cu-induced HM stress on overall photosynthesis performance, PI ABS and PI CS were determined in L. minor exposed to various intensities of Cu stress. Cu stress led to a significant effect on the performance index on absorption basis (PI ABS ) and performance index of PS II and PS I (PI CS ) in L. minor. PI ABS and PI CS continuously increased with increasing concentration of Cu up to 30 µM, and then declined sharply with the progression of Cu-induced HM stress. The lowest performance index on absorption basis (PI ABS ) and performance index of PS II and PS I (PI CS ) were recorded in plants cultivated on media containing 40 µM Cu (Figs. 2C, 3). is longer than others in all quadrates. Thus, the higher concentration of Cu was significantly affecting the major JIP parameters located in quadrate II and III. The mild Cu stress up to 20 µM was less toxic as compared to severe stress and plants performed better which was described by performance index parameters in quadrate IV (Fig. 6).
In Table 3, values of the lethal dese responsible for 50% mortality (LD 50 ) and 90% mortality (LD 90 ), calculated by probit analysis with 95% probability level, are given. The LD 50

Discussion
Many studies on the plant's physiological changes under various HM stress have been reported. These studies indicated that plants have developed a series of mechanisms to protect themselves from these adverse environmental threats. ChlF analyses have been shown to detect complex biochemical alteration in photosynthetic     92,93 . The increased level of relative variable fluorescence under Cu treatment indicates that the electron transfer at the donor side of PSII was affected. The affected F V /F O can be due to the modified unquenchable fluorescence (F O ) that altered the energy relay from antenna complex to reaction center 94 . According to PCA analysis the quantum yield was positively correlated with the electron transport per cross-section while negatively correlated (Fig. 6) with F O /F M located in the opposite direction of the PCA loading www.nature.com/scientificreports/ plot, which was also confirmed by the Correlation matrix (Fig. 7). Another possibility of reduced maximum primary yield under Cu stress can be the substitution of central atom of chlorophyll molecule, Mg by Cu. This substitution can hinders photosynthetic light-harvesting in the affected chlorophyll molecules 95 .
The "JIP test" of fluorescence transient in photosynthetic organisms, subjected to abiotic stress revealed a marked decrease in φPo 96 . The slight reduction in φP O might be due to a decrease in PSII photochemical efficiency resulting from Cu stress (in most higher plants having usually a value in the range of 0.78-0.84 97 ). In the light condition, a reduction in the maximum quantum yield of PSII (φPo) shows that HM stress inhibits the redox reaction following Q A and causes a delay in electron transport between Q A − and Q B· 90 . These parameters are very important and provide relevant information on electron transport activity at the PSII acceptor sites. The present finding suggested that Cu treatment reduces the electron transport at the PSII acceptor site in L. minor.
Energy pipeline models (membrane and leaf model), presented in Figs (Figs. 6, 7). The ABS/RC is determined by taking the total amount of photons absorbed by Chl molecules throughout all RCs by the total number of active RCs 58 . The ratio of active/inactive RCs influences it, and as the number of active centers rose, the ABS/RC ratio reduced. TR O /RC is the maximum rate at which an exciton is captured by the RC, resulting in a decrease in Q A . An increase in this ratio indicates that all the Q A has been reduced 83 . Reduction in ET O /RC describes that the re-oxidation of reduced Q A through electron transport in an www.nature.com/scientificreports/ active RC is decreased because a greater number of the active RC are available, hence it only reflects the activity of active RCs. Figure 4 demonstrates a reduction in per active RC electron transport but an overall increase in electron transport. The ratio of total dissipation of un-trapped excitation energy from all RCs to the number of active RCs is defined as DI O /RC. Dissipation arises as heat, fluorescence, and energy transfer to other systems and the ratios of active/inactive RCs also have an impact. Due to the effective utilisation of energy by the active RCs, the ratio of total dissipation to the number of active RCs (DI O /RC) is not very impacted 102,103 . The F V /F M ratio = (F M − F O )/F M is an important JIP parameter that represents the conversion efficiency of primary light energy in the PS II reaction center and is used as a stress indicator in a large number of photosynthetic studies 53,82,87 . However, since it is dependent on F O and F M fluorescence levels, this quantitative parameter is not usually sensitive enough to detect alteration across samples. Srivastava et al. employed the performance index (PI), a novel, more responsive, and significant parameter to measure photosynthesic efficiency under stress 104 . The performance index, PI, is derived using three (or four) components based on reaction center density, trapping efficiency, and electron transport efficiency, in the same way as a Goldman equation 105 . As a result, if any of these components is affected by stress, the effect will be visible in the performance index, which has a higher sensitivity. Performance index (PI ABS ) is calculated on an energy absorption basis while the performance index on cross-section (PI CS ) is obtained by multiplying the performance index on absorption basis PI ABS , by the phenomenological energy flux, ABS/CS = Fo (or F M ): and the value of PIabs and P CS significantly lowered in a plant grown under Cu stress (Fig. 2C). PI ABS are decreased due to reduced activity of the RC so the overall activity of the RC is decreased 41 (Table 3) demonstrated that this molecule can be considered as highly toxic to L. minor. Copper phytotoxicity was assessed through the visible symptoms of toxicity and determination of the concentration that results in a 50% reduction in the growth of L. minor (LD 50). According to Teisseire & Guy (2000), CuSO4 at 10 μM was inhibitory for L. minor (Teisseire and Guy). However, some plant species tolerate this element at concentrations higher than those used in medium cultures. Our study indicated that, L. minor was sensitive to copper for concentrations ≥ 25 µM 108 . This is caused by the different duckweed species used and by the different test conditions, especially concerning the nutrient media as well as by the methods of evaluation (Appenroth et al. 109 ).

Conclusion
In the present study, the efficacy of ChlF kinetics in the detection of Cu-induced HM stress was analysed in L. minor. Treatment of lower concentration of Cu (0.0-20.0 µM) had mild negative effect on photosynthesis. As the Cu is an essential micronutrient and plays a vital role in many biochemical processes, hence under moderate metal concentration the L. minor performed normally without any deleterious effect. A typical OJIP curve was obtained which shows that the plant efficiently used the solar energy for photosynthesis which is expressed in the term of increased active reaction center and performance index. In contrast, at higher Cu concentration (30.0-40.0 µM), the OJIP curve has been flattened due to a reduction in electron transport towards PSI (P 700 ), and a major portion of absorbed energy was dissipated in the form of heat because of an increased number of the inactive reaction center. Conclusively, phenomenological energy flux (ABS/CS M , TR O /CS M and ET O /CS M ), maximum quantum yield (φP O ), performance indexes (PI ABS and PI CS ) are powerful indicators of HM stress in plants and can be used for rapid detection of HM-induced water pollutant.Additionally, the key OJIP parameters screened in this paper could be a good tool for the rapid detaction of primary mode of action of HM on the photosynthetic apparatus in L. minor.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.